Coupling of an antibody to one or more functional groups, residues or moieties may add one or more desired properties or functionalities to the antibody. Desired properties may include the possibility to attach the antibody to a solid phase, the introduction of a marker (such as e.g. an enzyme, biotin, gold particles, etc.), increase of the half-life, the solubility and/or the absorption of the antibody and/or attachment of a drug. Coupling of an antibody to one or more functional groups, residues or moieties may also reduce undesired properties such as reduction of the immunogenicity and/or the toxicity of the antibody. For a further description of possible modifications and/or groups, residues or moieties that might improve the properties of an antibody, references is also made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).
Chemical coupling of antibodies with drugs, radioisotopes, proteins or other molecules has been widely investigated (see e.g. Luttmann et al. Immunology, The Experimenter Series, Elsevier pp. 30-39; King D. J. Applications and Engineering of Monoclonal Antibodies, Taylor & Francis Books Ltd. ISBN: 0748404228, pp. 58-66; McCafferty et al. Antibody Engineering, A practical approach. Ed. Hames B. D., IRL Press) and a range of chemical approaches are available. Chemical conjugation is the only option for the attachment to antibodies of non-proteinaceous materials such as drugs and polyethylene glycol.
A variety of amino acid residues on the surface of the antibody molecule can be used for chemical modification and coupling such as tyrosine, aspartic and glutamic acid, lysine and cysteine. In the case of cysteine, antibody molecules do not normally contain a free thiol group as all of the available cysteine residues form disulphide bonds. In addition, loss of some or all of the antigen binding ability of the antibody is a common consequence of non-specific conjugation methods which may result from the modification of amino acids close to the antigen binding site. This is even more pronounced when antigen binding fragments are used. Therefore, functional groups are preferably attached to cysteines via a suitable linker or spacer. These linkers may provide an additional thiol group and might reduce the loss of functionality of the antibody due to sterical hindrance.
One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as polyethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). The conjugation of the PEG to the target protein can be effected in a number of different ways (see e.g. Zalipsky 1995, Bioconjug. Chem. 6: 150-165; Greenwald et al. 2000, Crit. Rev. Ther. Drug Carrier Syst. 17: 101-161). Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Chapman et al. 1999, Nat. Biotechnol. 17: 780-783; Yang et al. 2003, Protein Eng. 16: 761-770). For example, PEG may be attached to a cysteine residue that naturally occurs in the protein, the protein may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of the protein and/or one or more cysteine residues may be provided within a linker segment (Yang et al. 2003, Protein Eng. 16: 761-770; EP 1 160 255, WO 01/94585, EP 1 639 011).
Nanobodies (as further described herein) are characterized by formation of the antigen binding site by a single variable domain, which does not require interaction with a further domain (e.g. in the form of VH/VL interaction) for antigen recognition. Production of Nanobodies, in lower eukaryotic hosts such as Pichia pastoris has been extensively described in WO 94/25591. Attachment of functional groups, residues or moieties, such as polyethylene glycol, to a Nanobody has been described e.g. in WO 08/101985, WO 08/142164, WO 09/068625, WO 09/068627 and WO 08/020079.